Confocal laser scanning microscopy (CLSM) was used to localize the major allergen,Bet vI in birch pollen. Pollen grains ofBelula pendulawere collected from catkins that had begun to release their pollen, freeze dried, and stored at 4°C. The pollen material was cryosectioned and stained with indirect immunofluorescence using the monoclonal antibody Bv 10. The observed localization ofBet vI was dependent on the treatment of the grains. When they had been prefixed with gaseous formaldehyde followed by acetoneBet vI was found in the cytoplasm. When the prefixation was omitted, a minor portion ofBet vI also appeared in the exine in the aperture regions. The allergen was not observed in the intine or the Zwischenkörper. Our suggestion is that the normal route for excretion ofBet vI is via the apertures on contact between pollen and the stigmatic surface of the pistil.